1. Field of the Invention
This invention relates to a flavin reductase gene from a luminescent bacterium Vibrio fischeri, a flavin reductase enzyme, a recombinant vector containing the gene and a bacterium containing the recombinant vector.
2. Description of the Related Art
An enzyme luciferase from a luminescent bacterium forms an oxidized flavin mononucleotide (hereinafter referred to as FMN) and a long chain fatty acid, using as substrates, a reduced flavin mononucleotide, (hereinafter referred to as FMNH.sub.2) and a long chain fatty aldehyde and in the presence of the enzyme, and at that time, catalyzes a blue color-luminescence reaction.
In the cell, the substrate FMNH.sub.2 used in this reaction is supplied from a reduced nicotinamide adenine dinucleotide : flavin mononucleotide (NADH : FMN) reductase and a reduced nicotinamide adenine dinucleotide phosphate: flavin mononucleotide (NADPH : FMN) reductase, and the long chain fatty aldehyde is supplied from a fatty acid reductase complex.
Recently, Spyrou et al. isolated a flavin reductase gene from Escherichia coli and clarified its primary structure and discloses it in [Spyrou, G., Haggard-Ljungquist, E., Krook, M., Jornvall, H., Nilsson, E. & Reichard, P. (1991), J. Bacteriol. 173, 3673-3679].
The present inventors have also already isolated an FMN reductase gene of a luminescent bacterium, Vibrio fischeri and have determined its nucleotide sequence. Further, we have succeeded in expressing the gene in Escherichia coli (Japanese patent application No. Hei 03-351,717). Further, we have isolated a flavin reductase gene from a luminescent bacterium, Xenorhabdus luminescens and have succeeded in its expression in Escherichia coli (Japanese patent application No. Hei 04-279029). This time, we have isolated a novel flavin reductase gene distinct from the above one (the contents corresponding to the present application).
FMNH.sub.2 is easily and instantaneously autoxidized in air and converted into FMN. In order to make the luminescence ability of the bacterial luciferase exhibit up to the most, it is necessary to always continuously supply the FMNH.sub.2 as the substrate, and FMN reductase is most important for attaining the object.
As described above, this enzyme couples with the bacterial luciferase and catalyzes the reaction of converting FMN as the product of the luciferase reaction into FMNH.sub.2 ; hence when the luciferase is made coexistent with FMN reductase in the reaction system, it is possible to maintain the luminescence reaction. Namely, since the bacterial luciferase turns over many time, luminescence continues as far as a large excess of a long chain aldehyde is present in the reaction system.
As described above, FMN reductase is inevitable for making the best use of the bacterial luciferase, and when its gene is obtained, it is possible to prepare the present enzyme in a large quantity.
Problem to Be Solved by the Invention
The present inventors have made extensive research, and as a result, have isolated a flavin reductase gene from a luminescent bacterium, Vibrio fischeri (ATCC 7744), and have succeeded in clarifying its primary structure, and further have succeeded in preparing Escherichia coli expressing the gene in a large quantity. Thus, the present invention has been completed.
Namely, in view of the above technical situation, the object of the present invention is to provide a flavin reductase gene of luminescent bacterium, Vibrio fischeri, and a flavin reductase, and the object is further to provide a recombinant vector containing the gene and a bacterium containing the recombinant vector.